CN103911445B - A kind of AS-PCR primer design method, gene pleiomorphism detecting method and test kit - Google Patents
A kind of AS-PCR primer design method, gene pleiomorphism detecting method and test kit Download PDFInfo
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Abstract
The present invention relates to a kind of AS-PCR primer design method, gene pleiomorphism detecting method and test kit, described method and test kit are used for detecting to determine whether allele variant exists to the target sequence that may comprise allelic variation region.Described AS-PCR primer design method comprises the following steps: (i) for the target sequence design AS-PCR primer comprising allelic variation region to be measured; (ii) the AS-PCR primer (i) designed to step is modified, and makes 3 ' of described AS-PCR primer to hold last base with the group that can shield described AS-PCR primer and directly extend.Compared to the detection site ability that traditional AS-PCR is single, gene pleiomorphism primer design method of the present invention and detection method and test kit can fuzzy diagnosis mutational sites, and probe and primer can be united two into one, primer synthesis cost, design time are saved, design also facilitates understandable, easily grasps.
Description
Technical field
The invention belongs to diagnostic nucleic acid reagent field, be specifically related to a kind of allele-specific polymerase chain reaction (AS-PCR) primer design method.The invention still further relates to a kind of AS-PCR gene pleiomorphism detecting method and test kit, described method and test kit are used for (namely suspecting) that the target sequence comprising allelic variation region detects to determine whether allele variant exists.
Background technology
Allele specific oligonucleotide analytical method (allele-specific oligonucleotide, ASO) is the mutation detection techniques based on hybridization, conventional to detect known mutations.The oligonucleotide fragment of design one section of such as 15 ~ 20bp, wherein contain the position that known mutations occurs, as probe, when hybridizing with the sample DNA be fixed on film, difference due to a base can cause Tm value decline 5 ~ 7.5 DEG C, therefore by strictly controlling hybridization conditions, can identify in sample DNA whether there is sudden change.
The allele-specific polymerase chain reaction (AS-PCR) combined by ASO and PCR is the method that current one is usually used in analyzing gene polymorphism, particularly single nucleotide polymorphism (SNP).Polymorphism (polymorphism) refers in a biotic population, there is two or more discontinuous anomalies or genotype (genotype) or allelotrope (allele), also known as genetic polymorphism (genetic polymorphism) or gene pleiomorphism simultaneously and often.Inherently, polymorphism results from the variation on gene level, generally occurs in the region of not proteins encoded in gene order and does not have the region of important regulatory function.Single nucleotide polymorphism (SNP), the difference of the single base be namely dispersed in, comprises the disappearance of single base, insertion and displacement, wherein in the majority with the situation of single base substitution.SNP is the class polymorphism received much attention at present, because it has the effect of genetic marker in the researchs such as genetic diseases of being everlasting, disease susceptibility, adverse drug reaction, therefore, it is detected as medical diagnosis on disease and early diagnosis, and pharmacology research etc. provides huge potential possibility.
In AS-PCR, one of primer of PCR or two primers are designed to anneal in the site of sequence variations, can distinguish the not isoallele of same gene, and these allelic gene types comprise the replacement of base, insertion and disappearance.AS-PCR make use of the fidelity of reproduction of archaeal dna polymerase: hold last base correctly to mate primer compared to 3 ', and 3 ' holds the primer of last base mispairing greatly to reduce (being generally 1/100 to 1/100,000) in extension efficiency.3 ' holds the primer of last base mispairing to cause thus extending difficulty, and pcr amplification reduces, and therefore distinguishes sudden change easily via detection.
The primer 3 ' of tradition AS-PCR holds last base crucial especially, is necessary for gene polymorphism sites.To detect SNP, its principle as shown in Figure 1.In Figure 1A, for target sequence design allele-specific primers (calling AS primer in the following text) 1-1,1-2 of comprising SNP site, namely, on Standard PCR design of primers basis, make SNP site to be measured just be positioned at the 3 ' end of AS primer 1-1,1-2, i.e. the base difference (respectively corresponding wild-type and possible saltant type) of primer 1-1 and 1-2 only 3 ' end, all the other base sequences are consistent, wherein 5 ' end X represents hydroxyl in base or phosphate, and 3 ' end Y represents the hydroxyl in base.In Figure 1B, 2-1 and 2-2 is respectively wild-type and the mutant DNA double-strand at allelotrope place, and wherein 2-1b and 2-2b is respectively the template strand matched with AS primer.As shown in Figure 1 C, the principle of holding last base strictly complementary to match according to 3 ', realizes allelic specific amplification.That is, if the template complete complementary in the AS primer in Figure 1A and Figure 1B matches, then form duplex structure shown in 3-1,3-2 in Fig. 1 C, PCR reaction is carried out smoothly, if form situation shown in 3-3,3-4 in Fig. 1 C, 3 ' terminal bases mispairing, then PCR reaction can not be carried out.By analyzing pcr amplification result, judge SNP situation, if primer 1-2 increase and primer 1-1 without obvious amplification, then show to there is described sudden change.
The requirement of tradition AS-PCR to archaeal dna polymerase is higher.The non-AS-PCR of PCR() archaeal dna polymerase conventional in reaction system has A type and Type B two kinds, and Aform DNA polysaccharase has 5 ' and holds the DNA polymerase activity held to 3 ', as Taq archaeal dna polymerase etc.; B-form DNA polysaccharase not only has 5 ' and holds the DNA polymerase activity held to 3 ', also have 3 ' and hold the DNA 5 prime excision enzyme activity held to 5 ', immediately can identify and excise the 3 ' base mismatch held (because mispairing causes tilting), thus the base of remaining non-mispairing is continued amplification, such as pfu archaeal dna polymerase, KOD archaeal dna polymerase, etc.Because traditional AS-PCR is just needing the mismatched primers utilizing pleomorphism site, so only Aform DNA polysaccharase can be used in amplified reaction, the B-form DNA polysaccharase that base mismatch can be excised can not be used.
Fig. 2 A and 2B schematically shows A type and the operative condition of B-form DNA polysaccharase in normal PCR system.Wherein, Fig. 2 A is the operative condition of Aform DNA polysaccharase in normal PCR system, when primer (3 ' end is hydroxyl) mates completely with template, under the effect of Aform DNA polysaccharase, primer is increased on (left side), and when primer 3 ' is held with template mispairing, under annealing temperature, mispairing part tilts, can not be combined with template, Aform DNA polysaccharase is inoperative, and primer can not increase (right side), utilize this characteristic, traditional AS-PCR is achieved the detection of polymorphic allele; Fig. 2 B is the operative condition of B-form DNA polysaccharase in normal PCR system, when primer mates completely with template, under the effect of B-form DNA polysaccharase, primer is increased (left side), and when primer 3 ' is held with template mispairing, mispairing part tilts, B-form DNA polysaccharase first plays 3 ' and holds the DNA excision enzyme effect of holding to 5 ', by mispairing Partial Resection, the primer of remaining non-mispairing is continued to increase on (right side) under archaeal dna polymerase effect, and obvious like this cannot being used for differentiates polymorphic allele in traditional AS-PCR.
There is following shortcoming in tradition AS-PCR technology:
The first, because AS primer will design for saltant type to be measured, so traditional AS-PCR technology only can detect known pleomorphism site, unknown mutation type can not be detected.
The second, traditional AS-PCR technology is in detection amplification, and one adopts electrophoresis analytical method, and this mode complicated operation is time-consuming, and cost is high, and technology is more difficult universal; Another kind adopts real-time fluorescence quantitative PCR, and this also needs extra complex sign to have the fluorescent probe of fluorophor and quenching of fluorescence group.
Therefore, there are the needs of the traditional AS-PCR technology of improvement in industry.
Summary of the invention
The present invention makes for the shortcoming of above-mentioned traditional AS-PCR just.
An objects and advantages of the present invention part will be listed in the following description, and another part is easy to those skilled in the art to be expected based on description below.
An object of the present invention is to provide a kind of method of design of AS-PCR primer, said method comprising the steps of: (i) for the target sequence design AS-PCR primer comprising allelic variation region to be measured; (ii) the AS-PCR primer (i) designed to step is modified, and makes 3 ' of described AS-PCR primer to hold last base with the group that can shield described AS-PCR primer and directly extend.The primer utilizing aforesaid method of the present invention to design can be used for determining that whether different from the known type AS-PCR of allelotrope to be measured reacts, such as distinguishing wild-type and saltant type, and different from traditional AS-PCR design of primers, consider that allelic concrete mutation type to be measured is no longer necessary.
Another object of the present invention is to provide a kind of AS-PCR gene pleiomorphism detecting method, described method is used for detecting to determine whether allele variant exists to the target sequence that may comprise allelic variation region, it is characterized in that said method comprising the steps of: (i) for the described target sequence design AS-PCR primer that may comprise allelic variation region; (ii) the AS-PCR primer (i) designed to step is modified, and makes 3 ' of described AS-PCR primer to hold last base with the group that can shield described AS-PCR primer and directly extend; (iii) use target sequence described in (ii) the modified AS-PCR primer pair of step to carry out pcr amplification, described pcr amplification uses B-form DNA polysaccharase; (iv) analytical procedure amplification (iii) determines whether described allele variant exists.
In above-mentioned AS-PCR primer design method and/or AS-PCR gene pleiomorphism detecting method, because designed AS-PCR primer 3 ' end has the group that can shield primer and directly extend, when with template DNA in conjunction with time, if primer and template complete complementary, because 3 ' of primer has carried out going hydroxylation to modify, cannot with dNTP polymerization reaction take place, therefore primer does not extend.Otherwise, if primer and template mispairing, namely not exclusively complementary, then during annealing, the tilting of mispairing part is not combined with template, now 3 ' end of B-form DNA polysaccharase works to 5 ' end 5 prime excision enzyme activity, primer 3 ' is held the Partial Resection of the not complementary pairing tilted, 3 ' terminal hydroxy group of residue primer portion exposes (after the phosphodiester bond on DNA chain between dNTP is hydrolyzed under the effect of B-form DNA polysaccharase, primer 3 ' is held and is produced hydroxyl, 5 ' end of dNTP or the DNA chain under hydrolysis forms phosphate), so, enzyme cut after primer can at archaeal dna polymerase effect downward-extension.
Whether therefore, contrary with traditional AS-PCR, AS-PCR primer of the present invention only just extends when mispairing, does not then extend time complementary with template strand, so just can not distinguish mutation type and detect polymorphic allele and exist.Specifically, if there is multiple possible sudden change in a certain allelotrope, such as single or multiple base is inserted, lacks or is replaced, and people only need detect sample whether exist sudden change and concrete mutation type need not be determined time, then can only for wild-type design AS-PCR primer, if amplification shows that sample exists sudden change, otherwise do not increase and show that sample is wild-type.Than traditional AS-PCR primer, it extends and mispairing occurs then does not extend when complementary with template strand, therefore will design primer for each sudden change, and the advantage of method of the present invention is apparent.Certainly, primer design method of the present invention does not get rid of the detection for concrete mutation type yet.
In AS-PCR primer design method of the present invention and/or AS-PCR gene pleiomorphism detecting method, preferably, step (i) described in allelic variation region be arranged in 1st ~ 6 bases that 3 ' of described AS-PCR primer holds, the length of described primer is 18bp ~ 30bp.Make B-form DNA polymerase enzyme cut identified region as far as possible near primer 3 ' end like this, and digested after still there is enough chain lengths, in order to avoid enzyme cut after primer too short and easily to come off from template strand, reduce amplification efficiency.
Preferably, 5 ' of AS-PCR primer described in amplified reaction end is made to the Tm value in described allelic variation region at 48 DEG C ~ 52 DEG C.Along with Tm increases, primer and template mispairing probability increase, along with Tm reduce, primer from template come off probability increase.
In AS-PCR primer design method of the present invention and/or AS-PCR gene pleiomorphism detecting method, the modification that step is (ii) held described AS-PCR primer 3 ' includes but not limited to that dehydroxylation, hydroxy position modify amino, hydroxy position modifies carboxyl, hydroxy position modifies sulfydryl or hydroxy position modifies phosphate, etc.More preferably, step is (ii) modified to mark fluorescent quenching group to the hydroxy position that described AS-PCR primer 3 ' is held, and step (ii) comprises 5 ' the end mark fluorescent group to described AS-PCR primer further.Like this, the AS-PCR primer of mark directly can be used as the probe of real-time fluorescence PCR, realizes uniting two into one of primer and probe.Mark the described fluorophor that described AS-PCR primer 5 ' holds to include but not limited to: FAM, VIC, HEX, ROX, Taxas Red or CY5, etc., mark the described fluorescent quenching group that described AS-PCR primer 3 ' holds to include but not limited to: BHQ1, BHQ2, BHQ3, Dabcyle, Temra, Eclipse, etc.Above-mentioned fluorophor and quenching of fluorescence group and marking method thereof are known in the art.
Fig. 3 A and 3B schematically shows the action principle of B-form DNA polysaccharase in AS-PCR system of the present invention.In Fig. 3 A, primer 3 ' end is connected with the group Y ' of shielding primer extension, and in Fig. 3 B, primer 5 ' end is connected with fluorophor X
0, 3 ' end is connected with quenching group Y
0.When primer mates completely with template, due to 3 ' terminal hydroxy group conductively-closed, primer cannot increase (left side), and when primer 3 ' holds base and template mispairing, under annealing temperature, mispairing part tilts, B-form DNA polysaccharase first plays 3 ' and holds the DNA excision enzyme effect of holding to 5 ', and by mispairing Partial Resection, the primer of remaining non-mispairing is continued to increase (right side) under archaeal dna polymerase effect.
In AS-PCR gene pleiomorphism detecting method of the present invention, step described B-form DNA polysaccharase (iii) includes but not limited to pfu archaeal dna polymerase, KOD archaeal dna polymerase, etc.Described B-form DNA polysaccharase is known in the art, and can obtain from open channel, and such as, pfu archaeal dna polymerase can be purchased from TAKARA; KOD archaeal dna polymerase can be purchased from TOYOBO.
Another object of the present invention is to provide a kind of AS-PCR genetic polymorphism detection test kit, wherein at least comprises following composition: the step of the above-mentioned AS-PCR gene pleiomorphism detecting method of a. (i)-(ii) designed AS-PCR primer; B. the step of the above-mentioned AS-PCR gene pleiomorphism detecting method (iii) middle B-form DNA polysaccharase used.
Compared to the detection site ability that traditional AS-PCR is single, sudden change identification primer design method of the present invention and detection method and test kit can fuzzy diagnosis mutational sites, namely use wild-type as abrupt climatic change sequence, and need not consider concrete mutation type.During the variation of this people's oncogene in complexity detects, as the sudden change of more than 40 kinds that Human epidermal growth factor receptor (EGF-R ELISA) gene the 19th exon occurs, or one of subclass of people C-KIT(Tyrosylprotein kinase) sudden change of gene the 11st exon 2 more than 5 kinds, only need to design one or several wild type marker primer corresponding with mutantional hotspot regional sequence.Therefore saved primer synthesis cost, design time compared to traditional AS-PCR, design also facilitates understandable, easily grasps.
Another advantage of the present invention primer and probe can be united two into one, achieve the real-time detection of AS-PCR, compared to hybridization or the electrophoresis detection mode of traditional AS-PCR, decrease operation steps, and stopped pipe detects, the possibility that after greatly reducing, PCR primer is polluted; And compared to traditional real-time fluorescence AS-PCR probe and primer separately designed, the present invention also greatly reduces synthesis cost.
Accompanying drawing explanation
Can become apparent, to being described as follows of institute's accompanying drawing for allowing above and other object of the present invention, feature, advantage and embodiment:
Fig. 1 schematically shows the principle that traditional AS-PCR detects SNP;
Fig. 2 A-2B schematically shows A type and the operative condition of B-form DNA polysaccharase in PCR system, and wherein, Fig. 2 A is the operative condition of Aform DNA polysaccharase in PCR system, and Fig. 2 B is the operative condition of B-form DNA polysaccharase in PCR system;
Fig. 3 A and 3B schematically shows the action principle of B-form DNA polysaccharase in AS-PCR system of the present invention;
Fig. 4 A and 4B is the typical amplification figure detecting heterozygous and the homozygous sample obtained according to the inventive method.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly, below in conjunction with the drawings and the specific embodiments, the present invention is further elaborated.Should be appreciated that embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
embodiment 1. people Methylene tetrahydrofolate reductase (MTHFR) gene pleiomorphism AS-PCR detects:
The concrete steps condition of PCR can with reference to the record in " Molecular Cloning: A Laboratory guide (third edition) " (the 8th chapter " polymerase chain reaction amplification in vitro DNA ", 597-701 page, Science Press, in August, 2002).
1.1. people's mthfr gene polymorphism AS-PCR design of primers:
People MTHFR-01 is for detecting SNP(GTCTGCGGGAG A/C CGATTTCAT) site T allelotrope, people MTHFR-02 is for detecting SNP(GTCTGCGGGAG A/CCGATTTCAT) site C allelotrope, people MTHFR-03 be downstream primer.
People MTHFR-01:5'-AAGGAGAAGGTGTCTGCGGGAGC – 3'
People MTHFR-02:5'-AAGGAGAAGGTGTCTGCGGGAGT – 3'
People MTHFR-03:5'-gaaagatcccggggacgatg-3'
1.2. Modify to primer:
People MTHFR-01:5'(FAM)-AAGGAGAAGGTGTCTGCGGGAGC – (BHQ1) 3'
People MTHFR-02:5'(VIC)-AAGGAGAAGGTGTCTGCGGGAGT – (BHQ1) 3'
1.3. templated synthesis:
Synthesis people MTHFR sequence 1 is as template DNA (italic thickened portion is primer binding sequence):
CTTCATCCCTCGCCTTGAACAGGTGGAGGCCAGCCTCTCCTGACTGTCATCCCTATTGGCAGGTTACCCCAAAGGCCACCCCGAAGCAGGGAGCTTTGAGGCTGACCTGAAGCACTTGAAGGAGAAGGTGTCTGCGGGAGCCGATTTCATCATCACGCAGCTTTTCTTTGAGGCTGACACATTCTTCCGCTTTGTGAAGGCATGCACCGACATGGGCATCACTTGCCCCATCGTCCCCGGGATCTTTCCCATCCAGGTGAGGGGCCCAGGAGAGCCCATAAGCTCCCTCCACCCCACTCTCACCGCACCGTCCTCGCACAG
The sequence 2 of synthesis people MTHFR is as template DNA:
CTTCATCCCTCGCCTTGAACAGGTGGAGGCCAGCCTCTCCTGACTGTCATCCCTATTGGCAGGTTACCCCAAAGGCCACCCCGAAGCAGGGAGCTTTGAGGCTGACCTGAAGCACTTGAAGGAGAAGGTGTCTGCGGGAGTCGATTTCATCATCACGCAGCTTTTCTTTGAGGCTGACACATTCTTCCGCTTTGTGAAGGCATGCACCGACATGGGCATCACTTGCCCCATCGTCCCCGGGATCTTTCCCATCCAGGTGAGGGGCCCAGGAGAGCCCATAAGCTCCCTCCACCCCACTCTCACCGCACCGTCCTCGCACAG
1.4. dosing: according to concentration preparation below reaction solution: 2mM dNTP, 2.5mM Mg
2+, 1U/ reacts pfu archaeal dna polymerase, 10mM pH8.3Tris, 50mM KCl, 0.1pmol/ul primer people MTHFR-01,0.1pmol/ul primer people MTHFR-02,0.1pmol/ul primer people MTHFR-03, template DNA, adding water to volume is 20ul.
The template DNA of sequence 1 and sequence 2 has carried out (1pg/ul, 10pg/ul, 100pg/ul, 1000pg/ul) after gradient dilution respectively, increases for fluorescent PCR.
Template consumption: ddH
2o(that is 0), 2pg/ reaction, 20pg/ reaction, 200pg/ reaction, 2000pg/ reaction.
1.5. the loop parameter used: 95 degrees Celsius 5 minutes
(95 degrees Celsius 10 seconds 58 degrees Celsius 45 seconds) 10 circulations
(95 degrees Celsius 10 seconds 60 degrees Celsius 45 seconds) 35 circulations
1.6. fluorescence quantitative PCR detection: use Bio-rad CFX96 fluorescent PCR instrument.
1.7. result: the Ct value that concentration is respectively the sample of 0pg/ul, 1pg/ul, 10pg/ul, 100pg/ul, 1000pg/ul sees the following form 1.
Table 1
"/" represents negative sample, without this SNP site; " N " represents that detected result is negative; Template add-on (sequence 1+ sequence 2) refers to that MTHFR sequence 1 and MTHFR sequence 2 put into the amount of PCR reaction.
1.8. result decision method: if Ct(VIC, C) and Ct(FAM, T) being all greater than 35, then interpret sample concentration is low or have suppression, need reform.If Ct(VIC, C) or Ct(FAM, T) in have a Ct value to be less than 32, then judge by following result:
I), Ct(VIC, C)-Ct(FAM, T) <-3, then judge that SNP site is that CC is homozygous;
Ii), Ct(VIC, C)-Ct(FAM, T) >3, then judge that SNP site is that TT is homozygous;
Iii) ,-1<Ct(VIC, C)-Ct(FAM, T) <1, then judge that SNP site is CT heterozygous.
Test confirms, each sample detected result is all consistent with order-checking background; Wherein sample 1 result is negative, sample 2 for SNP site be the homozygous sample of C, sample 3 for SNP site be the homozygous sample of T, sample 4 and 5 is heterozygous sample.See the typical amplification curve that Fig. 4, Fig. 4 A is homozygous sample, one of its two primers have amplification; Fig. 4 B is the typical amplification curve of heterozygous sample, and its two primers all have amplification.
embodiment 2. people TfR rs41295879 gene pleiomorphism design of primers and its application:
2.1. people TfR design of primers:
People TfR-01 is for detecting SNP(AAAATCC A/G GTGTAGGCA) site G allelotrope, people TfR-02 is for detecting SNP(AAAATCC A/G GTGTAGGCA) site A allelotrope, people TfR-03 be downstream primer.
People TfR-01:5'-CCTGGAGCTGCAAAATCCAG – 3'
People TfR-02:5'-CCTGGAGCTGCAAAATCCGG – 3'
People TfR-03:5'-CACCAATATTCAAAAGAATCAA-3'
2.2. Modify to primer:
People TfR-01:5'(FAM)-CCTGGAGCTGCAAAATCCAG – (BHQ1) 3'
People TfR-02:5'(VIC)-CCTGGAGCTGCAAAATCCGG – (BHQ1) 3'
2.3. templated synthesis:
Synthesis people TfR sequence 1 is as template DNA:
AGCTGATTATATAAGTTCTTTCTTCCTCTTTTTTCTTTTTTAAAGATCACTATGTTGTAGTTGGGGCCCAGAGAGATGCATGGGGCCCTGGAGCTGCAAAATCCAGTGTAGGCACAGCTCTCCTATTGAAACTTGCCCAGATGTTCTCAGATATGGTCTTAAAAGGTAGAGTACAAATTTTGATTCTTTTGAATATTGGTGCACTGCATACAGTTCTAGATGTTATACTGTGCTTTGCTCACTTTGCCTGCATTCCTGTGGTTCTCATGCTAGACTCTAGTTCTTAAATACAAGGCAGATT
Synthesis people TfR sequence 2 is as template DNA:
AGCTGATTATATAAGTTCTTTCTTCCTCTTTTTTCTTTTTTAAAGATCACTATGTTGTAGTTGGGGCCCAGAGAGATGCATGGGGCCCTGGAGCTGCAAAATCCGGTGTAGGCACAGCTCTCCTATTGAAACTTGCCCAGATGTTCTCAGATATGGTCTTAAAAGGTAGAGTACAAATTTTGATTCTTTTGAATATTGGTGCACTGCATACAGTTCTAGATGTTATACTGTGCTTTGCTCACTTTGCCTGCATTCCTGTGGTTCTCATGCTAGACTCTAGTTCTTAAATACAAGGCAGATT
2.4. dosing: according to concentration preparation below reaction solution: 2mM dNTP, 2.5mM Mg
2+, 1U/ reacts KOD archaeal dna polymerase, 10mM pH8.3Tris, 50mM KCl, 0.1pmol/ul primer TfR-01,0.1pmol/ul primer people TfR-02,0.1pmol/ul primer people TfR-03, template DNA, adding water to volume is 20ul.
Wherein the template DNA of sequence 1 and sequence 2 has carried out (1pg/ul, 10pg/ul, 100pg/ul, 1000pg/ul) after gradient dilution respectively, increases for fluorescent PCR.
Template consumption: ddH
2o, 2pg/ reaction, 20pg/ reaction, 200pg/ reaction, 2000pg/ reaction.
2.5. the loop parameter used: 95 degrees Celsius 5 minutes
(95 degrees Celsius 10 seconds 58 degrees Celsius 45 seconds) 10 circulations
(95 degrees Celsius 10 seconds 60 degrees Celsius 45 seconds) 35 circulations
2.6. quantitative real time PCR Instrument instrument: Bio-rad CFX96 fluorescent PCR instrument.
2.7. result: the Ct value that concentration is respectively the sample of 0pg/ul, 1pg/ul, 10pg/ul, 100pg/ul, 1000pg/ul sees the following form 2.
Table 2
"/" represents negative sample, without this SNP site; " N " represents that detected result is negative; Template add-on (sequence 1+ sequence 2) refers to that people TfR sequence 1 and people TfR sequence 2 put into the amount of PCR reaction.
2.8. result decision method: if Ct(VIC, A) and Ct(FAM, G) being all greater than 35, then interpret sample concentration is low or have suppression, need reform.If Ct(VIC, A) or Ct(FAM, G) in have a Ct value to be less than 32, then judge by following result:
I), Ct(VIC, A)-Ct(FAM, G) <-3, then judge that SNP site is that AA is homozygous;
Ii), Ct(VIC, A)-Ct(FAM, G) >3, then judge that SNP site is that GG is homozygous;
Iii) ,-1<Ct(VIC, A)-Ct(FAM, G) <1, then judge that SNP site is AG heterozygous.
Test confirms, each sample detected result is all consistent with order-checking background; Wherein sample 1 result is negative, sample 2 for SNP site be the homozygous sample of A, sample 3,5 for SNP site be the homozygous sample of G, sample 4 is heterozygous sample.
embodiment 3. people TfR rs3817672 gene pleiomorphism design of primers and its application:
3.1. people TfR design of primers:
People TfR-04 is for detecting SNP(ACAGACTTCACC A/G GCACCATCAAGT) site G allelotrope, people TfR-05 is for detecting SNP(ACAGACTTCACC A/GGCACCATCAAGT) site A allelotrope, people TfR-06 be downstream primer.
People TfR-04:5'-GGACAGCACAGACTTCACCAGC – 3'
People TfR-05:5'-GGACAGCACAGACTTCACCGGC – 3'
People TfR-06:5'-CCACAGTGTCACCATTATTGTTTC-3'
3.2. Modify to primer:
People TfR-04:5'(FAM)-GGACAGCACAGACTTCACCAGC – (BHQ1) 3'
People TfR-05:5'(VIC)-GGACAGCACAGACTTCACCGGC – (BHQ1) 3'
3.3. templated synthesis:
Synthesis people TfR sequence 3 is as template DNA:
ACTTCCCTGCAGCACGTCGCTTATATTGGGATGACCTGAAGAGAAAGTTGTCGGAGAAACTGGACAGCACAGACTTCACCAGCACCATCAAGTGAGTGCCAGCTGCTGTGCAAGTATCTAGACAAGTAATTCAAGAATTATGATAGGCCACGGGGAAACAATAATGGTGACACTGTGGGGAATGGCTTGTTAGAGAAGACAAGACTTGTCATGTTTAGCTAAAGAGGGGAAGGGGCCTGTAAAAAACTGAAACTATACTGG
Synthesis people TfR sequence 4 is as template DNA:
ACTTCCCTGCAGCACGTCGCTTATATTGGGATGACCTGAAGAGAAAGTTGTCGGAGAAACTGGACAGCACAGACTTCACCGGCACCATCAAGTGAGTGCCAGCTGCTGTGCAAGTATCTAGACAAGTAATTCAAGAATTATGATAGGCCACGGGGAAACAATAATGGTGACACTGTGGGGAATGGCTTGTTAGAGAAGACAAGACTTGTCATGTTTAGCTAAAGAGGGGAAGGGGCCTGTAAAAAACTGAAACTATACTGG
3.4. dosing: according to concentration preparation below reaction solution: 2mM dNTP, 2.5mM Mg
2+, 1U/ reacts KOD archaeal dna polymerase, 10mM pH8.3Tris, 50mM KCl, 0.1pmol/ul primer people TfR-04,0.1pmol/ul primer people TfR-05,0.1pmol/ul primer people TfR-06, template DNA, adding water to volume is 20ul.
Wherein template DNA has carried out (1pg/ul, 10pg/ul, 100pg/ul, 1000pg/ul) after gradient dilution respectively, increases for fluorescent PCR.
Template consumption: 0/reaction, 2pg/ reaction, 20pg/ reaction, 200pg/ reaction, 2000pg/ reaction.
3.5. the loop parameter used: 95 degrees Celsius 5 minutes
(95 degrees Celsius 10 seconds 58 degrees Celsius 45 seconds) 10 circulations
(95 degrees Celsius 10 seconds 60 degrees Celsius 45 seconds) 35 circulations
3.6. quantitative real time PCR Instrument instrument: Bio-rad CFX96 fluorescent PCR instrument.
3.7. result: concentration is respectively ddH
2the Ct value of O, 1pg/ul, 10pg/ul, 100pg/ul, 1000pg/ul sees the following form 3.
Table 3
"/" represents negative sample, without this SNP site; " N " represents that detected result is negative; Add-on (sequence 3+ sequence 4) refers to that people TfR sequence 3 and people TfR sequence 4 put into the amount of PCR reaction.
3.8. result decision method: if Ct(VIC, A) and Ct(FAM, G) being all greater than 35, then interpret sample concentration is low or have suppression, need reform.If Ct(VIC, A) or Ct(FAM, G) in have a Ct value to be less than 32, then judge by following result:
I), Ct(VIC, A)-Ct(FAM, G) <-3, then judge that SNP site is that AA is homozygous;
Ii), Ct(VIC, A)-Ct(FAM, G) >3, then judge that SNP site is that GG is homozygous;
Iii) ,-1<Ct(VIC, A)-Ct(FAM, G) <1, then judge that SNP site is AG heterozygous.
Test confirms, each sample detected result is all consistent with order-checking background; Wherein sample 1 result is negative, sample 2,4 for SNP site be the homozygous sample of G, sample 3 for SNP site be the homozygous sample of A, sample 5 is heterozygous sample.
Embodiment 4. Human epidermal growth factor receptor genovariation design of primers and its application:
The present embodiment does not distinguish the abrupt climatic change of mutation type for the contingent various mutations in mutantional hotspot district of Human epidermal growth factor receptor gene the 19th exon, wherein have chosen 64 kinds of mutation types, and synthesis template sequence is verified.
4.1. Human epidermal growth factor receptor design of primers:
Human epidermal growth factor receptor-01 for detect SNP site mutator gene, Human epidermal growth factor receptor-02 for detecting total EGFR gene quantity, Human epidermal growth factor receptor-03 is downstream primer.(in table 4, E-1 is EGFR wild-type DNA-sequence to SNP type, and E-2 ~ E-65 is the gene order of undergoing mutation, and be wherein with underscore to be the region of genetically deficient, italic is sequence area distinguishing with wild-type after transgenation)
The mutation type of table 4:EGFR
| E-1 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GAA TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-2 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GAA TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-3 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GUA TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-4 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG AUU CCA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-5 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG AUC CCC AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-6 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG AUA CCG AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-7 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG AAA TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-8 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GAU CCT AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-9 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GAC CCT AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-10 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG CAC CCT AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-11 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG CAA CCT AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-12 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG CGA CCT AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-13 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG CGT CCT AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-14 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG CGC CCT AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-15 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG CGG CCT AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-16 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG AGA CCT AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-17 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG AGG CCT AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-18 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GUN GCN AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-19 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG ATN CCN AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-20 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GUN CCN AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-21 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG AUN UCN AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-22 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG TTN UCN AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-23 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG CTN UCN AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-24 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GTN UCN AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-25 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG AUN AGN AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-26 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG TTN AGN AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-27 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG CTN AGN AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-28 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GTN AGN AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-29 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG CAA TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-30 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG CAG TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-31 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GUA TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-32 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GUC TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-33 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GUT TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-34 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GUG TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-35 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG CCA TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-36 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG CCC TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-37 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG CCG TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-38 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG CCT TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-39 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GAA TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-40 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG TCA TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-41 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG TCC TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-42 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG TCG TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-43 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG TCT TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-44 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG AGT TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-45 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG AGC TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-46 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG AUA TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-47 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG AUC TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-48 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG AUT TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-49 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG UUT TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-50 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG UUC TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-51 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GGN TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-52 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GAT TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-53 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GAC TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-54 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GTN TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-55 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GCN TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-56 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG AUA TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-57 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GCN TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-58 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GAT TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-59 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GAC TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-60 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GUN TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-61 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GAA TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-62 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GAA TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-63 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GAA TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-64 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GAA TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-65 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GAA TTC AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
Human epidermal growth factor receptor-01:5'-AAATTCCCGTCGCTATCAAGGAATTA – 3'
Human epidermal growth factor receptor-02:5'-GTGAGAAAGTTAAAATTCCCGTA – 3'
Human epidermal growth factor receptor-03:5'-ATCGAGGATTTCCTTGTTG-3'
4.2. Modify to primer:
Human epidermal growth factor receptor-01:5'(FAM)-AAATTCCCGTCGCTATCAAGGAATTA – (BHQ1) 3'
Human epidermal growth factor receptor-02:5'(VIC)-GTGAGAAAGTTAAAATTCCCGTA – (BHQ1) 3'
4.3. templated synthesis:
Synthesis Human epidermal growth factor receptor sequence E-1 ~ E-65 is as template DNA:
| E-1 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GAA TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-2 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-3 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GUA TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-4 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG AUU CCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-5 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG AUC CCC ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-6 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG AUA CCGACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-7 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG AAA TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-8 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GAU CCT ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-9 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GAC CCT ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-10 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG CAC CCT ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-11 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG CAA CCT ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-12 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG CGA CCT ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-13 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG CGT CCT ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-14 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG CGC CCT ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-15 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG CGG CCT ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-16 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG AGA CCT ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-17 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG AGG CCT ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-18 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GUN GCN TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-19 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG ATN CCN TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-20 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GUN CCN CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-21 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG AUN UCN CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-22 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG TTN UCN CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-23 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG CTN UCN CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-24 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GTN UCN CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-25 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG AUN AGN CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-26 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG TTN AGN CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-27 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG CTN AGN CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-28 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GTN AGN CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-29 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG CAA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-30 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG CAG ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-31 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GUA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-32 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GUC ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-33 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GUT ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-34 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GUG ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-35 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG CCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-36 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG CCC ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-37 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG CCG ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-38 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG CCT ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-39 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-40 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG TCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-41 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG TCC ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-42 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG TCG ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-43 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG TCT ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-44 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG AGT ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-45 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG AGC ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-46 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG AUA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-47 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG AUC TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-48 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG AUT TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-49 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG UUT TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-50 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG UUC TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-51 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GGN TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-52 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GAT TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-53 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GAC TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-54 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GTN TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-55 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GCN TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-56 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG AUA CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-57 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GCN CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-58 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GAT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-59 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GAC CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-60 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GUN CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-61 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG TTA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-62 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-63 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-64 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG CCG AAA GCCAACAAGGAAATCCTCGAT |
| E-65 | GGTGAGAAAGTTAAAATTCCCGTCGCTATC AAG GAA AGA GAA GCA ACA TCT CCG AAA GCCAACAAGGAAATCCTCGAT |
4.4. dosing: react KOD archaeal dna polymerase, 10mM pH8.3Tris, 50mM KCl, 0.1pmol/ul primer Human epidermal growth factor receptor-01,0.1pmol/ul primer Human epidermal growth factor receptor-02,0.1pmol/ul primer Human epidermal growth factor receptor-03, template DNA according to concentration preparation below reaction solution: 2mM dNTP, 2.5mM Mg2+, 1U/, adding water to volume is 20ul.
Wherein template DNA has carried out (1pg/ul, 10pg/ul, 100pg/ul, 1000pg/ul) after gradient dilution respectively, increases for fluorescent PCR.
Template consumption: ddH
2o, 2pg/ reaction, 20pg/ reaction, 200pg/ reaction, 2000pg/ reaction.
4.5. the loop parameter used: 95 degrees Celsius 5 minutes
(95 degrees Celsius 10 seconds 58 degrees Celsius 45 seconds) 10 circulations
(95 degrees Celsius 10 seconds 60 degrees Celsius 45 seconds) 35 circulations
4.6. quantitative real time PCR Instrument instrument: Bio-rad CFX96 fluorescent PCR instrument.
4.7. result: the synthetic DNA getting 50pg/ul does template, the Ct value of detection sees the following form 4.
4.8. result decision method: if Ct(VIC, W) and Ct(FAM, M) being all greater than 35, then interpret sample concentration is low or have suppression, need reform.If Ct(VIC, W) or Ct(FAM, M) in have a Ct value to be less than 32, then judge by following result.
I), Ct(VIC, W)-Ct(FAM, M) <-6, then judge that EGFR is wild;
Ii), Ct(VIC, W)-Ct(FAM, M) >-6, then judge that EGFR undergos mutation.
Test confirms, each sample detected result is consistent with practical situation.Demonstrate according to method of the present invention, for the mutantional hotspot region of Human epidermal growth factor receptor gene, only adopt the AS-PCR primer for wild type site design, this various mutation type in transgenation hot spot region can be detected.
What deserves to be explained is, although above preferred implementation is carried out testing detect for the unite two into one fluorescent PCR (quenching of fluorescence group shields primer 3 ' terminal hydroxy group) that designs of probe and primer, but those skilled in the art can fully understand according to its background knowledge grasped and technician of the present invention, design of primers principle proposed by the invention is equally applicable to conventional electrophoretic and detects or the detection of traditional fluorescent PCR, this obtains experimental verification too, above lifted embodiment is owing to having the test operation step simplified the most, it is most preferred embodiment.
Therefore; the foregoing is only the preferred embodiment of the present invention; not in order to limit the present invention; the scope of protection of present invention limited by claims and its equivalent substitute mode; all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (9)
1. a method of design for AS-PCR primer, said method comprising the steps of:
I () is for the target sequence design AS-PCR primer comprising allelic variation region to be measured;
(ii) the AS-PCR primer designed by step (i) is modified, makes 3 ' of described AS-PCR primer to hold last base with the group that can shield described AS-PCR primer and directly extend,
Wherein, the PCR reaction of the modified AS-PCR primer that step (ii) obtains for using B-form DNA polysaccharase and carrying out, makes described modified AS-PCR primer extending with during template strand mispairing, and does not extend time complementary with template strand,
Wherein, described B-form DNA polysaccharase refers to that not only having 5 ' holds the DNA polymerase activity held to 3 ', also has the archaeal dna polymerase of 3 ' end to 5 ' the DNA 5 prime excision enzyme activity held.
2. an AS-PCR gene pleiomorphism detecting method, described method is used for detecting to determine whether allele variant exists to the target sequence that may comprise allelic variation region, it is characterized in that said method comprising the steps of:
I () is for the described target sequence design AS-PCR primer that may comprise allelic variation region;
(ii) the AS-PCR primer designed by step (i) is modified, make 3 ' of described AS-PCR primer to hold last base with the group that can shield described AS-PCR primer and directly extend;
(iii) described in the modified AS-PCR primer pair using step (ii) to obtain, target sequence carries out pcr amplification, and described pcr amplification uses B-form DNA polysaccharase;
(iv) amplification of analytical procedure (iii) determines whether described allele variant exists,
Wherein, described B-form DNA polysaccharase refers to that not only having 5 ' holds the DNA polymerase activity held to 3 ', also has the archaeal dna polymerase of 3 ' end to 5 ' the DNA 5 prime excision enzyme activity held.
3. method according to claim 1 and 2, wherein the region of allelic variation described in step (i) is arranged in 1st ~ 6 bases that 3 ' of described AS-PCR primer is held, and the length of described primer is 18bp ~ 30bp.
4. method according to claim 1 and 2, wherein makes 5 ' of AS-PCR primer described in amplified reaction end be 48 DEG C ~ 52 DEG C to the Tm value in described allelic variation region.
5. method according to claim 1 and 2, wherein 3 ' the modification of holding of step (ii) to described AS-PCR primer comprises dehydroxylation, hydroxy position modifies amino, hydroxy position modifies carboxyl, hydroxy position modifies sulfydryl or hydroxy position modifies phosphate.
6. method according to claim 1 and 2, wherein step (ii) to 3 ' of described AS-PCR primer hold be modified to mark fluorescent quenching group, and step (ii) comprises further to 5 ' of described AS-PCR primer end mark fluorescent group.
7. method according to claim 6, wherein mark described fluorophor that described AS-PCR primer 5 ' holds and be selected from the group that following material forms: FAM, VIC, HEX, ROX, Taxas Red or CY5, mark described fluorescent quenching group that described AS-PCR primer 3 ' holds and be selected from the group that following material forms: BHQ1, BHQ2, BHQ3, Dabcyle, Temra or Eclipse.
8. method according to claim 1 and 2, wherein said B-form DNA polysaccharase is pfu archaeal dna polymerase or KOD archaeal dna polymerase.
9. an AS-PCR genetic polymorphism detection test kit, wherein at least comprises following composition:
A. the AS-PCR primer designed by the method steps (i) according to any one of claim 2 to 8-(ii);
B. the B-form DNA polysaccharase used in the method steps (iii) according to any one of claim 2 to 8;
Wherein, described B-form DNA polysaccharase refers to that not only having 5 ' holds the DNA polymerase activity held to 3 ', also has the archaeal dna polymerase of 3 ' end to 5 ' the DNA 5 prime excision enzyme activity held.
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